Translation of the abstract (English)

The estimated number of persons living with the human immunodeficiency virus type 1 (HIV-1) worldwide in 2007 was 33.2 million, whereas over four million people became newly infected. Thus, to control the alarming spread of HIV-1, a vital need exists for developing an effective vaccine that would prevent individuals from infection. The immediate goal of HIV vaccine research is to design candidate ...

Translation of the abstract (English)

The estimated number of persons living with the human immunodeficiency virus type 1 (HIV-1) worldwide in 2007 was 33.2 million, whereas over four million people became newly infected. Thus, to control the alarming spread of HIV-1, a vital need exists for developing an effective vaccine that would prevent individuals from infection. The immediate goal of HIV vaccine research is to design candidate vaccines that cause the immune system to produce protective responses from both of its major arms � cellular immunity and neutralizing antibodies. The main mode of protection that is induced by most antiviral vaccines seems to be mediated by neutralizing antibodies. Even during early phases of infection, when the level of antibodies induced by active immunization or administered by passive immunization is low, antibodies can reduce the size of the infecting inoculum and neutralize or eliminate virions during the first rounds of replication. In the case of HIV-1, this type of inhibition is attributed to antibodies that target the virus glycoprotein Env, especially to antibodies that are specific for the external part gp120. One vaccine strategy inducing both major arms of the adaptive immune system, humoral and cellular immunity, is the application of plasmid DNA. However, previous immunizations demonstrated that HIV Env DNA vaccines are relatively inefficient at inducing strong humoral and cellular immune responses.

In this study, three different approaches were used to enhance the immunogenicity of Env expressed from a DNA vaccine: (i) use of RNA- and codon-optimized gene inserts to increase expression rates, (ii) test of different signalpeptides to maximize secretion efficiency of the translated gp120 proteins and (iii) fusion of three copies of the molecular adjuvant human C3d (hC3d3). The immunostimulatory effect of C3d depends on the binding to its receptor CD21, located on B cells and follicular dendritic cells, thereby leading to an improved activation of B cells and an enhanced presentation of the antigen.

The generated DNA vaccines encoding a secreted monomeric form of a C-clade gp120 and C-terminally fused to three copies of the human C3d sequence (hC3d3) achieved a high and long-lasting expression of gp120 proteins in 293T cells. Whereas signal peptide variations had no influence on gp120 release from transfected mammalian cells, the C3d fusion markedly impaired expression as well as secretion rates of gp120. A construct containing two stop codons between gp120 and hC3d3 confirmed that the reduced expression is not depending on the transfection efficiency. Furthermore, the luciferase activity of an endogenous luciferase gene correlated with the length of the translated gp120 constructs, suggesting that the junction of hC3d3 leads to a decreased secretion efficiency of the fusion proteins.

Moreover, the hC3d3 moiety of secreted gp120 fusion proteins was capable of binding specifically to its receptor CD21 on B-cells. Since human C3d is able to bind CD21 receptors of other species, it was possible to test the gp120hC3d3 fusion constructs in mice and rabbits.

The most potent DNA constructs were tested in BALB/c-mice and in rabbits for the induction of reactive antibodies and T-cell responses. Short and long term immunization protocols revealed that the compromised production of hC3d3-fused antigens resulted in low gp120-specific antibody titers. However, gp120 DNA vaccines induced comparable T-cell responses in mice irrespective of hC3d3. In summary, these data suggest that the adjuvant properties of C3d were not capable of compensating the negative effects of poor expression on the stimulation of gp120-specific immune responses.

To overcome the limitations of gp120hC3d3 in vivo expression new constructs were designed allowing presentation of Env and C3d proteins on the surface of virus-like particles (VLP). These VLP constructs are based on a codon-optimized Gag sequence as well as on Env and C3d sequences fused to a transmembrane region (TM) anchoring the Env and C3d proteins in the VLP membrane. The efficient expression and secretion of Env- and C3d-bearing VLP was confirmed after transfection in 293T cells. Stable cell lines expressing Env and C3d on their surface were generated to enable a high production of HIV-1 VLP in eukaryotic cells. Further in vivo studies are planned to investigate the immunogenicity of these VLP.

This study indicates that neither use of different signal peptides nor hC3d3 fusion improve secretion efficiency or immunogenicity of codon-optimized gp120 DNA constructs. However, with the new designed VLP constructs it may be possible to overcome the reduced secretion efficiency of gp120hC3d3 fusion proteins and consequently improving the immunostimulatory potential of C3d.